The invention relates to a specimen chamber for the liquid treatment of at least one specimen, in particular a throughflow chamber, for the liquid treatment of biological specimens, such as, for example, a throughflow chamber for detecting the presence of nucleic acids and proteins by means of gensonde hybridisation or by means of antibodies.
Genomes of animals and plants contain genes in the order of size of 105, which are expressed in a specific manner. Knowledge of the gene expression in the tissues which form an organism is of fundamental significance in the understanding of the physiology and patho-physiology of a living being. Interest pertains in the expression analysis in individual biological cells. This is achieved by in-situ hybridisation (hereinafter ISH), in combination with immune histochemical processes. In addition to this, in this connection the hybridisation and representation of DNA microarrays is also of interest.
The term ISH is understood to mean the hybridisation of the genomic DNA or DNA generated by RT-PCR, or RNA synthesised in vitro, and, on the other hand, the hybridisation of cellular mRNA. From the principle of ISH, which is today generally described in text books, and which was first described by M. L. Pardue, a process has now developed which is carried out in laboratories all over the world, with a large number of manually-performed individual stages. Major factors of the process are nucleic acid hybrid formation and the evidencing of the hybrids. For the formation of hybrids, the specimens, which include sections of tissue with plant or animal cells, are subjected to liquid reagents under precisely controlled temperature conditions and in accordance with predetermined time programmes. An ISH may comprise 50 individual stages, for example, in which situation, at each individual stage, the tissue specimen must, within a few seconds, be brought to a predetermined temperature in the range from, for example, 4xc2x0 C. to 100xc2x0 C. with a precision of xc2x11xc2x0 C., and subjected to predetermined reagents. Both with regard to the time expenditure and with regard to the precision of the performance of the process, the manual procedure used hitherto is no longer suited to the demands for the processing of large numbers of specimens (xe2x80x9chigh throughputxe2x80x9d processes), which may involve hundreds or even thousands of prepared specimens.
With the aim of automating the ISH, throughflow chambers have been developed which are designed to contain tissue specimens or microarrays and carry out their liquid processing. An example of a conventional throughflow chamber 10xe2x80x2, which is marketed by the company of Shandon Southern Instruments Ltd., Great Britain, is illustrated in FIG. 12 (prior art). The throughflow chamber 10xe2x80x2 is based essentially on a combination of a cover plate arrangement 20xe2x80x2 and a carrier plate 30xe2x80x2, in which situation the cover plate arrangement 20xe2x80x2 features on its intake side a hopper-shaped extension 21xe2x80x2 to take in the processing liquids. A tissue specimen is placed in the space between the cover plate arrangement 20xe2x80x2 and the carrier plate 30xe2x80x2, and fixed in position there. The combination of the cover plate arrangement 20xe2x80x2 and the carrier plate 30xe2x80x2 is located in a cassette (not shown). For the liquid processing, the individual reagents are introduced via the extension 21xe2x80x2. Although the throughflow chamber 10xe2x80x2 allows for automatic use using pipetting robots, it suffers from the following disadvantages, which excludes its use for the ISH described heretofore, or restricts it to specific and less demanding process protocols.
Due to the complicated structure of the chamber sections with the outer cassette, temperature of the conventional throughflow chamber cannot be controlled in a reproducable manner. In addition to this, with the conventional throughflow chamber according to FIG. 12, the cover plate arrangement 20xe2x80x2 consists of a plastic which is not form-resistant if used at temperatures above 60xc2x0 C. which occur during ISH. Accordingly, the reproducibility of the reagent throughflow is limited. In addition, the design of the cover plate arrangement 20xe2x80x2 and the chemical composition of the cover plate renders impossible the uniform flow of the solutions containing formamide, used in the ISH, with the result that a spatially inhomogeneous hybridisation reaction arises, in which, for example, air bubbles occur or tissue particles or specimens float around. The combination of plastic with glass materials, too, for the formation of the chamber walls, excludes the possibility of reproducible temperature control.
In general, none of the generally known specimen or throughflow chambers are suitable for the analysis of DNA microarrays, in particular with processes with high throughput. Even if the specimen chambers for tissue specimens used hitherto in the laboratory sector has been capable of thermostatic adjustment, automatic reagent exchange is impossible. The manual loading of specimens with manual pipettes, however, is unsuitable for processes with high throughput.
The object of the invention is to provide an improved specimen chamber for liquid treatment of specimens, of which the temperature control is reliable and reproducible, and which will be form-resistant even at repeated temperature adjustment, and which, in particular, will allow for automatic process treatment in the throughflow principle even at high specimen throughput (about 100 to 500 per passage).
The basic idea of the invention particularly consists of the provision of a specimen chamber consisting of a base plate and a carrier plate, which are separated from one another by a predetermined interval by spacer elements, and are held together by means of a frame arrangement. The base and carrier plates have, for preference, the same surface area. In the assembled state they form an aligned packing unit, as a result of which the interval between the plates forms a receptacle or accommodation for the specimen. Given the design of the specimen chamber as a throughflow chamber, the accommodation (cutout) is open to two mutually opposed sides for the formation of an intake or an outlet, and closed to the other sides in a fluid-tight manner. To achieve this, the spacer elements run in strip form between the two edges of the base plate and carrier plate, aligned relative to one another, between the intake and the outlet. The base plate tapers at the intake (the thickness of the plate is reduced towards the end of the plate), to form a reservoir between the base plate and the carrier plate.
In the frame arrangement, the base plate and the carrier plate are held together aligned to one another. To provide for mutual fixation of the plates in the frame arrangement, provision is made for a clamp device, which on the one hand encompasses the frame arrangement, and, on the other, encompasses the plates arranged in it, creating a uniform compression tension. Under the effect of the compression tension, the cutout formed between the base plate and the carrier plate for the specimen is closed laterally along the spacer element, in a liquid-tight manner. The frame arrangement is also equipped with a hang-up device, which is designed to bring the throughflow chamber to a contact platform of a temperature control device.
The base and carrier plates of the specimen chamber according to the invention consist for preference of the same material, or of similar materials with regard to their thermal properties. This improves the temperature control, avoids deformations, and also causes the flow properties of the treatment liquids flowing through to be uniform at the taking of the specimen. The plates are for preference made of glass or, depending on the application, of a temperature-resistant plastic or quartz glass.
A subject of the invention is also a temperature adjustment device (referred to as a thermoblock) with a large number of temperature-adjustable contact platforms arranged in rows and gaps. The contact platforms in the operating state have an almost vertical (e.g. 15xc2x0) setting, or a setting pivoted further away from the vertical by a predetermined angle (up to almost 90xc2x0), in which situation a profiled projection is provided at the upper end to accommodate the specimen chamber according to the invention. The projection interacts with the hang-up device of the specimen chamber in such a way that, in the suspended state, the specimen chamber is drawn because of its weight towards the contact platform and establishes good thermal contact with it.
The invention has the following advantages. The specimen chamber according to the invention is particularly well-suited for use as a throughflow chamber for the liquid treatment of biological specimens. The chamber has an especially simple modular design with essentially only two elements (the base plate and the carrier plate), which are clamped in the frame. The glass structure is resistant to the reagents used in each case, in particular against aqueous and organic solvents. The parts of the throughflow chamber, which can be easily separated without further ado by releasing the clamping device are, on the one hand, easily and rapidly assembled after the carrier plate has been loaded with a specimen, and can be separated from one another without further ado after the processing of the specimen in order for the specimen to be removed and for the cleaning of the chamber. The throughflow chamber is capable of being treated in an autoclave, which is of advantage for reuse.
The compact design, exercising pressure tension by means of the clamping device, ensures optimum heat conduction from an outside temperature regulating device to the specimen in the throughflow chamber. The base and carrier plates, made of glass, do not undergo any deformation at all in the temperature ranges of interest, below 100xc2x0 C. The throughflow chamber can be rapidly located on the contact platform of a thermoplatform in a reproducible manner. At the same time, the frame arrangement of the chamber is constructed in such a way that the specimen remains accessible for visual observation and optical measurements.
A particularly important advantage is obtained if the carrier plate is formed by a standardised glass object carrier, such as is known from optical microscopy. The throughflow chamber according to the invention is thus rendered entirely compatible with the usual manipulation techniques for glass object carriers. In addition, the carrier plate can be designed with a substrate suitable for follow-on measurements or also for additional handling procedures. If the carrier plate consists, for example, of UV transmitting glass of optically high quality, the synthesis of oligonucleotides is made possible by masked photochemical reactions directly on the carrier plate.
The throughflow chamber according to the invention is characterised by substantially improved flow properties of the reagents thanks to the accommodation arrangement for the specimens. While, for example, in a conventional throughflow chamber according to FIG. 12 the processing liquid comes in contact with plastic on the one hand and with glass on the other, with the result that differences in the flow rates occur on both sides of the chamber, and therefore flow inhomogeneities or even bubbles form, with the throughflow chamber according to the invention the reagents which pass through encounter the same surface resistance on all the chamber walls, with the result that flow inhomogeneities or bubbles are avoided.
The use of the throughflow chamber according to the invention is not restricted to the ISH, but can be applied in general to any specimens desired, and biological specimens in particular, which are to be processed with liquid reagents, such as specimens for the examination of antibody reactions, for the hybridisation and representation of DNA microarrays, or similar functions.